Shaped wall geometry with dielectrophoretic and laser forces for particle separation and characterization

ABSTRACT

The combined value of integrating optical forces and electrokinetics allows for the pooled separation vectors of each to be applied, providing for separation based on combinations of features such as size, shape, refractive index, charge, charge distribution, charge mobility, permittivity, and deformability. The interplay of these separation vectors allow for the selective manipulation of analytes with a finer degree of variation. Embodiments include methods of method of separating particles in a microfluidic channel using a device comprising a microfluidic channel, a source of laser light focused by an optic into the microfluidic channel, and a source of electrical field operationally connected to the microfluidic channel via electrodes so that the laser light and the electrical field to act jointly on the particles in the microfluidic channel. Other devices and methods are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit as a divisional of U.S. patentapplication Ser. No. 15/183,531 filed Jun. 15, 2016 which in turn claimsthe benefit of U.S. patent application Ser. No. 14/043,384 filed on Oct.1, 2013, which in turn claims the benefit of U.S. ProvisionalApplication 61/709,290 filed on Oct. 3, 2012, the entirety of each ofwhich which is incorporated herein by reference.

BACKGROUND

A need exists for techniques for manipulation of analytes in liquids.

BRIEF SUMMARY

In one embodiment, a method of separating particles in a microfluidicchannel includes providing a device comprising a microfluidic channel, asource of laser light focused by an optic into the microfluidic channel,and a source of electrical field operationally connected to themicrofluidic channel via electrodes; flowing particles in a liquidthrough the microfluidic channel; and manipulating the laser light andthe electrical field to act jointly on the particles in the microfluidicchannel, thereby separating the particles based on size, shape,refractive index, electrical charge, electrical charge distribution,charge mobility, permittivity, and/or deformability.

In another embodiment, a method of separating particles in amicrofluidic channel includes a providing a device comprising a centralmicrofluidic channel operably connected to a first inlet and a secondinlet and a first outlet and a second outlet, and a source of laserlight focused by an optic into the central microfluidic channel in adirection orthogonal to the central microfluidic channel; flowingparticles and molecular species together in a first liquid through thefirst inlet while flowing a second liquid through the second inlet so asto pinch the flow from the first inlet; and applying optical force fromthe source of laser light to separate the particles from the molecularspecies such that the molecular species tend to exit the first outletand the particles tend to exit the second outlet.

In a further embodiment, a method of separating particles in amicrofluidic channel includes providing a device comprising amicrofluidic channel comprising an inlet and a plurality of exits, and asource of laser light focused by an optic to cross the microfluidicchannel at an angle; flowing a plurality of particles in a liquidthrough the inlet into the microfluidic channel; and selecting the angleof the laser light so as to produce an optical force on the particleswhile maximizing residence time in the laser light of selectedparticles, thus selectively separating the particles into the pluralityof exits.

In yet another embodiment, a method of separating particles in amicrofluidic channel includes providing a device comprising amicrofluidic channel configured to supply a dielectrophoretic (DEP)field to an interior of the channel via a (1) DEP electrode system or(2) insulator DEP system having shaped wall geometry or obstructiongeometry, and a source of laser light focused by an optic into themicrofluidic channel; flowing a plurality of particles in a liquid intothe microfluidic channel; and operating the laser light and DEP fieldjointly on particles in the microfluidic channel to trap the particlesor modify their velocity, wherein said DEP field is linear ornon-linear.

In a still further embodiment, a method of separating particles in amicrofluidic channel includes providing a device comprising amicrofluidic channel configured to supply a linear or non-lineardielectrophoretic (DEP) field to an interior of the channel via a (1)DEP electrode system or (2) insulator DEP system having shaped wallgeometry or obstruction geometry; flowing a plurality of particles in aliquid into the microfluidic channel; and operating the DEP field onparticles in the microfluidic channel to change velocity of theparticles, wherein said DEP field is linear or non-linear.

An embodiment of a device includes a microfluidic channel comprising aninlet and a plurality of exits, and a source of laser light focused byan optic to cross the microfluidic channel at a critical angle matchedto velocity of flow in the microfluidic channel so as to produce anoptical force on the particles while maximizing residence time in thelaser light of selected particles, thus separating the particles intothe plurality of exits, wherein the laser light is operable to applyforces to particles flowing through the microfluidic channel, therebyseparating the particles into the plurality of exits.

Another embodiment of a device includes a microfluidic channelconfigured to supply a linear or non-linear dielectrophoretic (DEP)field to an interior of the channel via a (1) DEP electrode system or(2) insulator DEP system having shaped wall geometry or obstructiongeometry, and a source of laser light focused by an optic into themicrofluidic channel, wherein the laser light and DEP field operatejointly on particles in the microfluidic channel to trap the particlesor modify their velocity.

A further embodiment of a device includes a microfluidic channelconfigured to supply a linear or non-linear dielectrophoretic (DEP)field to an interior of the channel via a (1) DEP electrode system or(2) insulator DEP system having shaped wall geometry or obstructiongeometry, wherein DEP field operates on particles in the microfluidicchannel to change velocity of the particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exemplary device layout.

FIG. 2 illustrates an embodiment wherein laser force is countered byfluid flow.

FIG. 3 illustrates an embodiment wherein laser force is co-directionalwith fluid flow.

FIGS. 4A through 4E illustrate embodiment having laser force orthogonalto fluidic flow. FIG. 4A shows a general schematic of continuousseparation. FIG. 4B illustrates the stage of sample introduction, withFIG. 4C showing a stage of a gated mode of operation with injection of aplug of charged molecular species and particles. FIG. 4D illustrates thebeginning of separation of molecular species from particles andsub-populations of molecular species from each other during gatedoperation, with FIG. 4E showing a moment later when such separation iscomplete.

FIG. 5 illustrates an embodiment of critical angle field flow.

FIG. 6 illustrates an embodiment of dielectrophoretic field flow with acounter-directionallaser.

FIG. 7 illustrates an embodiment of dielectrophoretic field flow with aco-directional laser.

FIGS. 8A and 8B illustrate embodiments of a dielectrophoretic (DEP) trapwith an orthogonal laser. FIG. 8A shows a side of a DEP trap while FIG.8B shows a cross-sectional view.

FIG. 9 illustrates one embodiment wherein an optical force acts onparticles in a DEP trap.

FIG. 10 illustrates a second embodiment wherein an optical force acts onparticles in a DEP trap.

FIG. 11 illustrates a shaped-wall embodiment employing a DEP trap.

FIG. 12 illustrates a shaped-obstruction embodiment employing a DEPtrap.

FIG. 13 illustrates an embodiment of DEP velocity modification.

FIG. 14 illustrates an embodiment of DEP velocity modification withoptical force deflection.

DETAILED DESCRIPTION

Definitions

Before describing the present invention in detail, it is to beunderstood that the terminology used in the specification is for thepurpose of describing particular embodiments, and is not necessarilyintended to be limiting. Although many methods, structures and materialssimilar, modified, or equivalent to those described herein can be usedin the practice of the present invention without undue experimentation,the preferred methods, structures and materials are described herein. Indescribing and claiming the present invention, the following terminologywill be used in accordance with the definitions set out below.

As used in this specification and the appended claims, the singularforms “a”, “an,” and “the” do not preclude plural referents, unless thecontent clearly dictates otherwise.

As used herein, the term “and/or” includes any and all combinations ofone or more of the associated listed items.

As used herein, the term “about” when used in conjunction with a statednumerical value or range denotes somewhat more or somewhat less than thestated value or range, to within a range of ±10% of that stated.

Description

The combined value of integrating optical forces and electrokineticsallows for the pooled separation vectors of each to be applied,providing for separation based on combinations of features such as size,shape, refractive index, charge, charge distribution, charge mobility,permittivity, and deformability. The interplay of these separationvectors allow for the selective manipulation of analytes with a finerdegree of variation. Thus, the larger portfolio of separation vectorspermits the probing of chemical composition, geometry, and internalstructure of analytes. In concert optical forces and electrokinetics canboth generate bulk fluid flow, e.g. electroosmotic flow, while alsoproviding a means of physically separating both particulate andmolecular species analyte mixtures, e.g. electrophoresis anddielectrophoresis. The technique also expands the separation andmanipulation of particulate and molecular species by working withouthaving to apply tag molecules to either type.

Experimental

An exemplary device layout is illustrated in FIG. 1. Of all thecomponents shown, two elements common to most (but not all) geometriesand configurations described herein include a source of laser light(130) as well as a source of electrical field (20). The laser source canrange in wavelengths from the UV to IR. The electrical sources can havea wide range of voltages in either DC (direct current), AC (alternatingcurrent), or mixed format. Each of these components are integrated intoa microfluidic platform constructed from glass, plastic, or othermaterials including combinations of different materials that enable theintroduction of laser light into the microfluidic chip (40), without theloss of significant portions of laser power or damage from absorbedwavelengths. The light from the laser source (80) can be directed intothe microfluidic chip via mirrors (120, 110) and focusing optics (90)held in place with optomechanical components (100) with the necessarydegrees of freedom to direct and align the laser into the microfluidicchip. The microfluidic chip is mounted on optomechanical components (10)that allow all necessary degrees of positional freedom. The electricalsource (20) is connected to the microfluidic device via electrodes (30,35). Fluid flow is fed to the microfluidic chip through several lengthsof tubing (70) from up to source volumes (50, 55, 60) and can beregulated with several pumping options including but not limited tosyringe pumps, peristaltic pumps, electroosmotic pumps, and pressurizedair over liquid pumps. This configuration allows for the elegantcombination and study of how optical, fluidic, electrophoretic and/ordielectrophoretic forces simultaneously interact and influence a sampleof interest within a custom microfluidic environment.

Counter Laser Field Flow

The combination of optical forces countered by flow, such aselectroosmotic flow (EOF), within a microfluidic device constitutes thetechnique known as counter laser field flow. Along the microfluidicchannel (160) or capillary the laser propagation (190, 210) is counteredby either pure EOF or by a combination of EOF and hydrodynamic flow(150). A detailed illustration of the configuration is shown in FIG. 2where interrogated samples (170, 180) are separated spatially (175)while being held in equilibrium between the fluidic, electrophoretic andoptical forces each sample experiences. Electric field is introduced viaelectrodes (140). Collimated laser light originates from a source (220)and is focused by an optic (200) into the microfluidic channel where theseparation occurs.

The system is capable of trapping and separating various sample types inthe channel on the basis of size, shape, refractive index and charge.Particle size can range from several tens of microns to hundreds ofnanometers. The addition of electrokinetics to the system of opticalchromatography allows for higher resolution separations by adding theability to probe particle charge as a separable factor.

Co-Directional Laser Field Flow

Detailed is a similar configuration to the Counter Laser Field Flowshown in FIG. 2; however, instead of the laser being directed counter tofluid flow the fluid flow and the direction of laser propagation are inthe same direction. In the exemplary illustration of FIG. 3, the laserpropagation (270, 290) in the microfluidic channel (250) or capillary iscountered by either pure EOF or by a combination of EOF and hydrodynamicflow (240). Interrogated samples (310, 300) become separated spatially(305), while being in equilibrium between the fluidic, electrophoreticand optical forces each sample experiences. Electric field is introducedvia electrodes (230). Collimated laser light originates from a source(260) and is focused by an optic (280) into the microfluidic channel(250), where the separation occurs.

Because the particles suspended in the flowing fluid travel in the samedirection as the laser propagation, this version of the device does nottrap the particulate matter delivered within the main flow. Instead, theposition of the particles added to the channel in a batch injection ordownstream are separated spatially in the channel and the largerparticles (310) equilibrate further downstream than the smallerparticles (300). Other samples of similarly sized particles or mixturesof any particles that share the same equilibrium position in a typicaloptical chromatography device may be separated based completely onsurface charge differences (surface charge density, polarity, density,surface area, roughness etc.), a capability not available in traditionaloptical chromatography.

Orthogonal Laser Field Flow

As seen in FIGS. 4A through 4E, a microfluidic device composed of laserbeam (360, 380) orthogonal to central channel (340) containing fluidflow produced by electroosmotic flow or hydrodynamic flow. From afluidic pinch sample flow (330) the device enables the separation of amixed sample stream (310) including molecular species and particleshaving various size/shape/refractive index. The device is capable tocontinuous high throughput separations when operated using a continuouslaser beam (360, 380) and fluid flow, shown in FIG. 4A. Sampleintroduction (310), pinch flow (330) from a secondary inlet (320), andoutlet separation (400 & 410) are generated by having multiple channelsconnect through a centralized separation/laser region (340). The numberof inlets and outlets can each be described by n+1 where n is a wholeinteger with a 1 or greater value. The interaction (370) of the opticalforce from the laser allows for the selective removal of particulatesfrom the molecular species stream (400) while also enabling selectivepositioning of the particles (410) to the various outlets. Collimatedlaser light originates from a source (350) and is focused by an optic(390) into the microfluidic channel (340), where the separation occurs.This system allows for both the separation of molecular species fromparticles as well as different particle types from each other. Particlescan be isolated from each other on the basis of size, shape, refractiveindex, and charge.

Additional operation modes include the gating of charged molecularspecies for separating positive or negative molecular components fromeach other and neutral components as well as particles. FIG. 4Billustrates the stage of sample introduction, with FIG. 4C showing astage of a gated mode of operation with injection of a plug of chargedmolecular species. FIG. 4D illustrates the beginning of separation ofmolecular species from particles during gated operation, with FIG. 4Eshowing a moment later when such separation is complete.

This preparative separation device can be mated to several traditionalanalytical detection schemes including but not limited to capillaryelectrophoresis, spectroscopic investigations, culturing, and antibodystudies.

Critical Angle Laser Field Flow

FIG. 5 illustrates a microfluidic device utilized to separate a broadsize range from tens of nm to several microns by continuously exposing amixed sized sample stream (420) to an angled continuous laser (490). Alaser light source (440) emits laser light (450, 490) that is focused byan optic (470). The angle of the laser (460) is matched to the velocityof the fluid flow (430), for example EOF, in such a manner that theamount of the optical force and the fluid velocity maintain the particlein the laser beam to maximize the residence time of the particle in thebeam. EOF is generated using an external electric field (410). A flatflow profile (430) generated via electroosmotic flow while a mixedsample is introduced with the laser beam situated at a critical anglepaired to the velocity of the fluid flow. The particles in the mixturewill predominantly move in the direction of the laser propagation. Giventhe critical angle of the beam the force directs the particles away fromthe sample stream in the same direction as the fluid flow.

As the scattering force scales with particle size and refractive index,larger/higher refractive indices particles will be maintained in thelaser beam longer than smaller/lower refractive indexed particles. Thecritical angle is determined to provide maximum displacement for thelargest/highest refractive indexed particle of the group to beseparated. Particles will exit the laser beam in a cascading pattern(480) predominately based on their interaction with the laser beam asthe electroosmotic flow is not a separative force, but electrophoreticbehavior will alter behavior depending on drag and the external fieldstrength. The mixed particle stream will be separated and enriched inseparate areas of the main channel (460) as they flow into separateexits (500) at the end of the channel allowing for enriched sub-samplesto be collected.

This embodiment is similar to the Counter Laser Field Flow methoddescribed above, but adds the element of dielectrophoresis (DEP) byshaping the channel to generate a non-uniform electric field (550 &710). This combination can be utilized to separate a mixed population ofparticles (540, 560, 690, & 700) via their DEP and optical properties.The optical force can be applied either counter to the EOF or pressureinduced flow (510 & 660) for the purposes of trapping or velocitymonitoring various particle types (540 & 560), or can be usedco-directionally with the EOF for sorting and velocity monitoring ofvarious types of particles (690 & 700). Electric fields (500 & 610) canbe DC or AC or mixtures of the two. DEP field can be any linear ornon-linear function of DEP generated by the channel (530 & 680). As seenin FIGS. 6 and 7, the laser is introduced to axially to the channel ineither a co-directional (630 & 650) or counter direction (570 & 590)with regard to fluid flow (510 & 660). Collimated laser light originatesfrom a source (600 & 620) and is focused by an optic (580 & 640) intothe microfluidic channel (520 & 670), leading to the shaped channelgeometry where the separation occurs.

Dielectrophoretic Trap Orthogonal Laser Manipulation & Separation

DEP traps (820) are utilized to generate populations of separatedparticles isolated on the basis of their electrokinetic properties(830), as illustrated in FIGS. 8 through 12. These separated populationsof particles can in turn be probed by optical forces (720, 740, 780,790, 860, 940) from a laser to explore other separation vectors, likerefractive index. Additionally, the optical force can be ramped in orderto gauge the strength of the trap, selectively “elute” heterogeneousparticles from the population (890 & 980), or to move the particles fromthe trap toward other channels (770 & 840) for further applications. Anexemplary device consists of an open microfluidic channel (750, 800,870, 970, 1020, 1070, 1110) constructed with a single or a series/arrayof DEP traps (810) that allow for fluid flow orthogonal to a laser (720& 780) that enters above trapped particle populations through a suitableportion of the channel wall (730, 960) and potential well or otherchannel structures (840) that may be positioned in an opposing channelwall (760, 850, 930, 1010). The DEP trap could be made through eitherinsulator or electrode based systems. Insulator DEP systems can generatenon-uniform field could be shaped wall geometries (880 & 1030) orobstruction geometries (950 & 1060) of any shape, e.g. round,elliptical, square, diamond, teardrop, and triangular columns.

The addition of the optical force allows for the controlled movement,manipulation, and separation of a plurality of particles (890 & 980)that have been trapped/collected in a DEP trap (880 & 950). Theselective movement of these particles based on size, shape, andrefractive index into an adjoining channel structure (910, 920, 990,1000) allows for further processing of the particles and collection foruse. From a vantage point above the microfluidic channel the shaped wall(1030) or the shaped obstructions (1060) with a straight side wall(1080) are visible as are the well or channel shunt features (1040 &1100) with the laser beam (1050 & 1090) outline also being visible overthe well/channel features at each structure.

Dielectrophoretic Velocity Monitoring For Identification And Sorting

As shown in FIG. 13, a microfluidic channel (1140 & 1160) generateseither a linear or non-linear DEP field (1190) that allows particles(1150) to pass through the middle of the channel while allowing theeffect of the field on the particles' velocity (acceleration,deceleration, or neutral) to be observed. The DEP field is generated viathe combination of an external electric field (1120) and the shapedchannel wall (1160). Fluid flow (1130) can be either EOF or pressureinduced flow, thus enabling the device to be manufactured in either EOFsupporting material or simply non-conductive materials. The effect onthe velocity of the particle allows the viewer to understand thepolarization, charge, and permeability of the particle to be determinedin a high-throughput fashion. The particle is view in the first regionof interest (ROI) (1170) and then again in the second ROI (1180) todetermine velocity. This system allows for identification, sorting, andmanipulation when combined with switching andcollection.Dielectrophoretic Velocity Monitoring for Cell Identificationand Sorting with Optical Force Deflection

Building on the previous embodiment, FIG. 14 illustrates a techniquewhereby adding an orthogonal optical force (1270) component allows thesystem to act like a 2-D gel, reporting information from DEP in additionto the refractive index from the optical force interaction (1280).Again, an external electric field (1200) generates a DEP field throughits being shaped by the geometry of the insulating channel wall (1220).Fluid flow (1210) can be either EOF or pressure induced flow, thusenabling the device to be manufactured in either EOF supporting materialor simply non-conductive materials. A source of laser light (1260)produces a laser beam (1250) which in turn is focused by an optic (1300)into the channel. After the particles (1230) are successively introducedto the channel (1220) via fluid flow (1210) the particle interacts withthe DEP field and their velocity is calculated as the particle passesthrough regions of interest (ROI) 1 and 2 (1240). Then the particlestream passes into the laser beam (1270) and are displaced from theiroriginal trajectory (1280). As the particle passes through ROI 3 (1290)the amount of displacement is quantified. This displacement isinformational as the particle's refractive index, shape, and size. Theplacement of the laser in the channel system can occur along curved andstraight portions of the channel.

Advantages and Applications

The possibility of separating chemically different particles offersimportant new possibilities for analysis and possible purifiedcollection of colloidal samples such as organic particulates, inorganicparticles (glass and metal particles), and biological species such ascells, bacteria, and viruses. Other samples may also be used, includingbut not limited to carbon nanotubes, quantum dots (including single,dimer & trimer forms), vesicles, organelles, samples relating toin-vitro fertilization (IVF), and liposomes.

These techniques may be used to distinguish and/or diagnose any numberof characteristics in samples, for example:

-   -   Live v dead organisms    -   Presence or absence of antibodies    -   Cell cycle stage    -   Blood cells types (e.g., red blood cells, white blood cells,        platelets)    -   Cancer    -   Infected cells, including cells infected with virus, bacteria,        or parasites (e.g., malaria or giardia)    -   Abnormal cells

The techniques may be used with, against, or neglecting gravity; in anarray; or in multiple passes.

The techniques may be used to distinguish many particles traits such as:

-   -   Shapes (including spherical or odd shapes)    -   Presence or absence of coatings    -   Absorbing and non-absorbing wavelength    -   Tagged and tag-less    -   Sorting particles of interest selectively from a plurality of        particles    -   Sorting particles from chemical or biochemical mixtures

Furthermore, as contemplated by one of skill in the art, it is possibleto use various laser types having any suitable beam geometry and type.

The systems described herein highlight the ability to separate particles(including but not limited to biologics such as cells) from chemical orbiochemical (e.g., protein or other) molecular species using a mixedoptical force and electrophoretic or dielectrophoretic forcecombination. This capability represents a significant leap forward in atechnology platform that could be used for combined chemical/biochemicaland biological analysis and sorting. The implications are far reaching,as a combined system capable of doing both chemical and biologicalwarfare agent detection does not yet exist.

Differentiation of biological samples such as bacteria is traditionallybased upon chemical differences in their capsules, membranes or othersurface or sub-surface features. Polysaccharides, lectins, lipoteichoicacids, and proteins are some of the biomolecules present in variousbacterial species and strains. It is well known that there exists asubstantial range of refractive indices in bacterial and viral samplesdue to their different chemical compositions. The ability to separatebiological species based upon physical and chemical properties usingonly light interaction with samples in a simple fluid flow is new andhas great potential benefits when applied to bio-warfare detection andbiomedical analysis. Not only are samples physically separable usinglight, but from their position in the separation field one can determinetheir unique intrinsic characteristics that will allow separation fromone another either actively or passively.

The ability to distinguish and sort one cell type from another ispredicated on force differences significant enough to generate physicalspace between the particles. In a microfluidic device, this generallymeans tens of microns of spatial separation are required at a minimum.Having an orthogonal forces working in tandem to effect a separation isadvantageous over either single force alone. While the electrophoreticforce is sensitive to surface charge, the optical force is sensitive tothe overall refractive index of the particle and indeed local opticalvariations within the cell or on the surface. Thus, the forces aresensitive to fundamentally different phenomena. The combination ofelectrophoretic or dielectrophoretic forces and optical force will be apowerful combination that will allow much finer separations to beachieved in a single instrument.

Other Techniques

Free from the requirements of chemical or immunological systems, atechnique based upon optical separation and detection alone willoutperform the current levels of performance achieved using othermethods. Systems based upon immunology require significant time and costoutlays to develop antibodies for new biological warfare (BW) agents(ref. 1). Furthermore, with these techniques, the detection of modifiedand/or unknown species in real time is not possible. Other techniquesbased upon DNA analysis, while very accurate, have problems includinglong analysis times, high cost and complexity, and delicateinstrumentation which is unlikely to be reduced in size significantly.Other methods of BW agent detection include fluorescence detection ofaerosols (ref. 2). Such techniques are limited in their ability todetect BW agents, as intrinsic fluorescence is derived from three aminoacids (with similar excitation and emission spectra) common to manybiological particles. Detection based upon fluorescence alone is notlikely to enable characterization of closely related species.

Field flow fractionation (FFF) is used for the separation of particulatematerials based upon their size, mass, density, charge, or otherphysical properties (refs. 3 and 4). The most basic variant of FFF usesa flow field to carry particulates in flow down a thin channel. Theseparticles are then subjected to a force (gravitational, flow,electrical, or other) that causes them to accumulate differentially atthe wall edge of the laminar flow field. Particles least affected by theapplied field will travel down the channel and exit sooner than thosethat are more effected by the applied field. Recently, strains of E.coli have been separated using a variation of FFF based upon thepresence or absence of fimbriae (ref. 5).

Flow cytometry is a technique used for characterizing cell populationswherein a sheath flow fluidic system hydrodynamically focuses the cellsinto a line (refs. 6 and 7). Once in a line, the stream containing thecells is interrogated by one or more laser beams of differingwavelength. Laser light scattering and laser induced fluorescence (fordye labeled particles or cells) measurements are made of the passingsamples. From these, many parameters can be determined including size,volume, granularity, and biochemical properties using dye labeled cellsurface antigens. In a cell sorting flow cytometer, after the opticalmeasurements, the sheath flow is vibrated at a high velocity creatingtiny droplets, which ideally contain only one cell. Depending on thecell type determined by the laser measurements, a charge is applied tothe droplets. When these charged droplets pass between two chargedplates they are deflected and can be collected, resulting in aspecifically directed separation.

While the above techniques enable separation, they suffer limitationsthat the current invention alleviates. Discrimination is not inherentlybased upon intrinsic chemical composition when using field flowfractionation. This limits the technique to essentially size basedseparation which are not as universally important as biochemicalspecificity when dealing with microbiological samples. With respect toflow cytometry, sorting can be achieved based upon physical propertiesand biochemical information derived from specific fluorescent probes.While being a powerful bioanalytical technique, flow cytometry suffersfrom the cost and complexity of the fluid system and the multiple colorlasers required to excite fluorescence in dye labeled biochemicalmarkers. More importantly, much biochemical specificity andidentification are achieved through the use of bioprobes, which bydefinition require prior knowledge for successful application. A methodsuch as optical laser separation which relies on the intrinsiccharacteristics of the biological species should prove more versatileand capable.

Optical trapping has been used for the repetitive sorting of particlesbased on their appearance, including size, shape, or other visiblefeatures. Recently, more sophisticated and automated optical techniqueshave been identified and developed to separate microscopic objects(refs. 9 and 10). These techniques involve arrays of optical traps in afluid flow to preferentially transport microscopic objects thatexperience a greater optical force away from those that experience alesser force.

Electrophoretic mobility has been used to sort cells, bacteria and otherparticles based upon size and charge ratios for some time (ref. 11).Electrophoresis is based on the surface charge of a particle or chargedmolecular species exposed to electrostatic forces generating a force onthe particle or molecular species that drags it toward the oppositelycharged pole of the field. This is described by the electrophoreticmobility (μe) being equal to the charge (q) divided by the viscous term(6rπη, r is the radius of the species, and η is the viscosity of themedium). The migration velocity (ve) of the species is derived bymultiplying μe by the electric field strength (E). This simpledescription is valid for molecular species. The charged walls of thefluidic device can also cause an additional electrokinetic phenomenaknown as electroosmotic flow (EOF) that generates bulk fluid flow.

Dielectrophoresis is a complex phenomenon where an electric fieldgradient interacts with dipoles and other multipoles properties ofmolecules and particles and elicits movement or trapping behavior (ref.12). The DEP was first described by Pohl in 1951, which presented atheoretical explanation for the force and detailed its use for removingsuspended particles from polymer solutions (ref. 13). Subsequently DEPhas been adapted and applied to a wide variety of biological structuressuch as cells, spores, bacteria, and viruses (ref. 14). Unlikeelectrophoresis, which acts primarily on the charge-to-size ratio of theparticle, DEP acts on a larger set of properties includingpolarizability, structure and medium permeability, and charge/chargedistribution. As a result higher resolution separations and greatersensitivity is often observed in DEP separations. Researchers using DEPhave achieved impressive results including separating cancer cells fromblood cells, (ref. 15) infected cells from normal cells, (ref. 16) andlive from dead cells (ref. 17).

Concluding Remarks

All documents mentioned herein are hereby incorporated by reference forthe purpose of disclosing and describing the particular materials andmethodologies for which the document was cited.

Although the present invention has been described in connection withpreferred embodiments thereof, it will be appreciated by those skilledin the art that additions, deletions, modifications, and substitutionsnot specifically described may be made without departing from the spiritand scope of the invention. Terminology used herein should not beconstrued as being “means-plus-function” language unless the term“means” is expressly used in association therewith.

REFERENCES

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The invention claimed is:
 1. A method of separating particles in amicrofluidic channel, the method comprising: providing a devicecomprising a microfluidic channel configured to supply a linear ornon-linear dielectrophoretic (DEP) field to an interior of the channelvia a (1) DEP electrode system or (2) insulator DEP system having shapedwall geometry or obstruction geometry; flowing a plurality of particlesin a liquid into the microfluidic channel; and operating the DEP fieldon particles in the microfluidic channel to change velocity of theparticles, wherein said DEP field is linear or non-linear and isgenerated at least in part by electrodes positioned at inlet and outletends of the channel and beyond a region of the microfluidic channelwhere particle sorting occurs.
 2. The method of claim 1, wherein theflow is electroosmotic and/or pressure driven.
 3. The method of claim 1,wherein the device further comprises a source of laser light focused byan optic into the microfluidic channel.
 4. The method of claim 3,wherein said laser light is focused axially in the same direction assaid flow.
 5. The method of claim 3, wherein said laser light is focusedaxially in the opposite direction as said flow.
 6. The method of claim3, wherein the flow is electroosmotic and/or pressure driven.
 7. Themethod of claim 3, wherein said device further comprises an adjoiningchannel structure, the method further comprising trapping populations ofsaid particles and focusing said laser light orthogonally to a flow axisof said microfluidic channel to elute one or more of said populationsinto the adjoining channel structure.